Breast Carcinoma Cell Line T47D Research Articles

Inhibition of Increased Invasiveness of Breast Cancer Cells With Acquired Tamoxifen Resistance by Suppression of CYR61.

Hormone sensitivity-targeted therapy with selective estrogen receptor modulators (SERMs), such as 4-hydroxytamoxifen (4-OHT), is the mainstay of treatment for breast cancers (BCs) that express estrogen receptor α (ERα). However, development of resistance limits this therapy approach. The question arises whether changes associated with 4-OHT resistance could be exploited therapeutically. First, 4-OHT-resistant sublines of ERα-positive breast carcinoma cell lines MCF-7 and T47D were generated. Viability was assessed by the Alamar Blue assay. Cell invasion was quantified in modified Boyden chambers with Matrigel. Changes in expression of CYR61, S100A4, and ERα were examined by RT-qPCR. Expression of CYR61 was suppressed by transient gene silencing using siRNA. Successful suppression was verified by western blot. Efficacy of 4-OHT treatment was analyzed by quantification of viability using Alamar Blue assay. Correlation of CYR61 levels in patients with luminal A BC to distant metastases-free survival was determined by Kaplan-Meier analysis. ERα-positive MCF-7 and T47D BC cells exhibit an extremely weak invasion rate. Acquired tamoxifen resistance significantly increased the invasive behavior of both tamoxifen-resistant MCF-7-TR and T47D-TR sublines. In addition, expression of CYR61 and S100A4 showed significantly increased levels, whereas expression of ERα was decreased. Suppression of CYR61 expression resulted in a significant decreased invasion rate. In addition, expression of S100A4 was reduced, whereas expression of ERα was increased. Furthermore, suppression of CYR61 resulted in re-sensitization to 4-OHT. High CYR61 levels in patients with luminal A BC resulted in reduced distant metastases-free survival. The prometastatic factor CYR61 appears to play an important role in the increased invasiveness of tamoxifen-resistant ERα-positive BC cells. Its suppression leads to a lower invasion rate. Given the few therapeutic options available for tamoxifen-resistant BC, therapy that reduces CYR61 may improve its treatability in future.

Inhibition of human ERα‐positive breast carcinoma cell by baicalein via cell cycle arrest and apoptosis

Baicalein is a naturally occurring flavone found in the four herbaceous plants: Scutellaria baicalensis, Scutellaria lateriflora, Oroxylum indicum, and the well‐known species Thymus vulgaris (thyme). Recently recognized as a potential anticancer therapeutic agent, baicalein is gaining popularity in cancer research laboratories. However, it is unclear whether baicalein can inhibit cancerous properties of the human ERα‐positive breast carcinoma cell line T47D. Here we demonstrate that baicalein can significantly inhibit cell growth and proliferation. Additionally, cell cycle analysis reveals that baicalein inhibits T47D breast cancer cell growth by inducing cell cycle arrest, suppressing colony formation and cell migration. Additionally, baicalein was shown to induce cell death by regulating genes and interacting with signaling pathways involved in apoptosis. These studies clearly suggest that baicalein has potent anti‐cancer properties and has great potential for cancer therapy.

Cytotoxicity Induced by Extracts of Pisolithus tinctorius Spores on Human Cancer and Normal Cell Lines—Evaluation of the Anticancer Potential

Fungi have been considered a potential source of natural anticancer drugs. However, studies on these organisms have mainly focused on compounds present in the sporocarp and mycelium. The aim of this study was to assess the anticancer potential of fungal spores using a bioassay-guided fractionation with cancer and normal cell lines. Crude extracts from spores of the basidiomycetous fungus Pisolithus tinctorius were prepared using five solvents/solvent mixtures in order to select the most effective crude extraction procedure. A dichloromethane/methanol (DCM/MeOH) mixture was found to produce the highest extraction yield, and this extract was fractionated into 11 fractions. Crude extracts and fractions were assayed for cytotoxicity in the human osteocarcinoma cell line MG63, the human breast carcinoma cell line T47D, the human colon adenocarcinoma cell line RKO, and the normal human brain capillary endothelial cell line hCMEC/D3. Cytotoxicity was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay. The results showed a reduction in cancer cell viability of approximately 95% with 4 of 11 fractions without a significant reduction in viability of hCMEC/D3 cells. Data demonstrated that spores of P. tinctorius might serve as an interesting source of compounds with potential anticancer properties.

Advanced Electrochemical in Vitro Detection of Superoxide Radicals with Fully Integrated Microsensor System

An electrochemical sensing system for in vitro measurement of superoxide radical is presented. The approach of the system is based on a fully integrated sensor setup in conventional tissue culture containment, utilizing novel direct oxidation of ROS on polymer covered gold microelectrode. The concept was shown to provide stable and sensitive measurement procedure without the need for enzymatic recognition elements. Sensor performance was investigated from artificial enzymatic superoxide production, with a sensitivity of 2247 AM-1m2 and outstanding high selectivity for main interfering substances. Online monitoring of superoxide release from human breast carcinoma cell line T-47D was successfully demonstrated.

Revealing the Anti-Tumor Effect of Artificial miRNA p-27-5p on Human Breast Carcinoma Cell Line T-47D

microRNAs (miRNAs) cause mRNA degradation or translation suppression of their target genes. Previous studies have found direct involvement of miRNAs in cancer initiation and progression. Artificial miRNAs, designed to target single or multiple genes of interest, provide a new therapeutic strategy for cancer. This study investigates the anti-tumor effect of a novel artificial miRNA, miR P-27-5p, on breast cancer. In this study, we reveal that miR P-27-5p downregulates the differential gene expressions associated with the protein modification process and regulation of cell cycle in T-47D cells. Introduction of this novel artificial miRNA, miR P-27-5p, into breast cell lines inhibits cell proliferation and induces the first “gap” phase (G1) cell cycle arrest in cancer cell lines but does not affect normal breast cells. We further show that miR P-27-5p targets the 3′-untranslated mRNA region (3′-UTR) of cyclin-dependent kinase 4 (CDK4) and reduces both the mRNA and protein level of CDK4, which in turn, interferes with phosphorylation of the retinoblastoma protein (RB1). Overall, our data suggest that the effects of miR p-27-5p on cell proliferation and G1 cell cycle arrest are through the downregulation of CDK4 and the suppression of RB1 phosphorylation. This study opens avenues for future therapies targeting breast cancer.

Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53

Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X L, Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.

Unique resistance of breast carcinoma cell line T47D to TRAIL but not anti-Fas is linked to p43cFLIPL

The majority of breast cancer cell lines are resistant to tumor necrosis factor -related apoptosis inducing ligand (TRAIL) induced apoptosis. TRAIL and Fas receptor death-inducing signaling complex (DISCs) formation are similar and involve ligand-dependent recruitment of FADD and caspase-8. We have found that the breast carcinoma cell line T47D is an unusual example of selective sensitivity to anti-Fas mAb treatment but resistant to TRAIL. Therefore, a detailed comparison of these two signaling pathways in one cell line should provide insight into the mechanism of TRAIL resistance. We observed that only anti-Fas mAb induces caspase activation and cell death in T47D. Further, FADD and caspase-8 interact with both TRAIL-R1 and TRAIL-R2, and that the amount of caspase-8 recruited by Fas-, TRAIL-R1 and TRAIL-R2 are the same. cFLIP(S) and cFLIP(R )isoforms block death receptor-induced apoptosis by inhibiting caspase-8 activation at the DISC; the role of cFLIP(L )at the DISC is still controversial. It has been suggested that the presence of the cleaved form of FLIP(L)-p43 at the DISC prevents caspase-8 cleavage. We found that both TRAIL and anti-Fas mAb-induced DISCs contain the cleaved form of p43 cFLIP(L) and its amount at the Fas DISC was higher compared to the TRAIL DISC. We also found that inhibition of cFLIP(L) expression in T47D cells decreased Fas-mediated caspase-8 activation and activation of effector caspases. We propose that in T47D p43 cFLIP(L) in the Fas-DISC may promote caspase-8 activation. The mechanism by which different amounts of p43cFLIP(L) regulates caspase-8 activation remains to be investigated.

Oncostatin M induces cell detachment and enhances the metastatic capacity of T-47D human breast carcinoma cells

Oncostatin M (OSM), an IL-6 family cytokine, has previously been shown to increase migration of several breast cancer cell lines in vitro. Our studies report additional effects of OSM treatment on the human breast carcinoma cell line T-47D. OSM treatment alters T-47D cell morphology from a normal epithelial phenotype to a mesenchymal-like phenotype that is associated with cell detachment from substratum. These effects are also seen with H3922 human breast cancer cells. OSM treatment of T-47D cells for 5–8 days leads to a three-fold increase in cell detachment. OSM-induced detachment of T-47D cells is blocked by the protein kinase inhibitors UO126 and bisindolylmaleimide, indicating a role for MAP kinases and protein kinase C in OSM signaling events that regulate cell detachment. T-47D cells induced to detach by OSM have a reduced capacity to re-adhere to laminin in comparison to other extracellular matrix components. Detached multi-cell aggregates of T-47D cells are viable, whereas detached single cells appear apoptotic. In addition, OSM treatment induces the secretion of the lysosomal proteases cathepsins D and L from T-47D cells, which have been implicated in invasion and metastasis. Importantly, OSM-treated T-47D cells show a 250% increase in invasive capacity as measured by the Matrigel invasion chamber assay. Collectively, these data demonstrate that OSM induces a motile/invasive phenotype in T-47D cells in vitro, and suggest that OSM may enhance metastasis in vivo. Our results suggest that OSM itself may be a valid therapeutic target.

Camptothecin Induces Nuclear Export of Prohibitin Preferentially in Transformed Cells through a CRM-1-dependent Mechanism

Prohibitin is a growth-suppressive protein that has multiple functions in the nucleus and the mitochondria. Our earlier studies had shown that prohibitin represses the activity of E2F transcription factors while enhancing p53-mediated transcription. At the same time, prohibitin has been implicated in mediating the proper folding of mitochondrial proteins. We had found that treatment of cells with camptothecin, a topoisomerase 1 inhibitor, led to the export of prohibitin and p53 from the nucleus to the mitochondria. Here we show that the camptothecin-induced export of prohibitin occurs preferentially in transformed cell lines, but not in untransformed or primary cells. Cells that did not display the translocation of prohibitin were refractive to the apoptotic effects of camptothecin. The translocation was mediated by a putative nuclear export signal at the C-terminal region of prohibitin; fusion of the nuclear export signal (NES) of prohibitin to green fluorescence protein led to its export from the nucleus. Leptomycin B could inhibit the nuclear export of prohibitin showing that it was a CRM-1-dependent event driven by Ran GTPase. Confirming this, prohibitin was found to physically interact with CRM-1, and this interaction was significantly higher in transformed cells. Delivery of a peptide corresponding to the NES of prohibitin prevented the export of prohibitin to cytoplasm and protected cells from apoptosis. These results suggest that the regulated translocation of prohibitin from the nucleus to the mitochondria facilitates its pleiotropic functions and might contribute to its anti-proliferative and tumor suppressive properties.

Comparison of Proteomic and Genomic Analyses of the Human Breast Cancer Cell Line T47D and the Antiestrogen-resistant Derivative T47D-r

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.

Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells.

We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer.

Cathepsin D Specifically Cleaves the Chemokines Macrophage Inflammatory Protein-1α, Macrophage Inflammatory Protein-1β, and SLC That Are Expressed in Human Breast Cancer

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.

Prolonged progestin treatment induces the promoter of CDK inhibitor p21 Cip1,Waf1 through activation of p53 in human breast and endometrial tumor cells

Progestins are frequently used in the treatment of advanced breast and endometrial cancer. The human breast carcinoma cell line T47D shows a biphasic response to progestins. Short-term progestin treatment leads to enhanced DNA synthesis, while this line is growth inhibited upon prolonged exposure. An important protein involved in growth regulation by progestins in this cell is the CDK inhibitor p21 Cip1,Waf1. We show that after 1 day of progestin treatment in T47D cells, the p21 promoter-proximal region containing Sp1 binding sites is crucial in the induction by progestins. However, after 3 days the activity of the promoter-distal region becomes predominant in T47D cells or the endometrial carcinoma cell line ECC1. This is dependent upon two domains within this region that contain p53 response elements. In ECC1 and T47D cells 3-day progestin treatment induces a reporter containing a p53 response element, but not a mutated version. This induction is due to activation of p53 by progestin, which may be caused by nuclear translocation of p53. These data indicate that upon prolonged exposure, progestins activate p53, in human breast and endometrial tumor cells, which up-regulates the p21 Cip1,Waf1 promoter. This may be an important mechanism involved in progestin-inhibited cellular proliferation in these cells.

Initiation of the breakage-fusion-bridge mechanism through common fragile site activation in human breast cancer cells: the model of PIP gene duplication from a break at FRA7I.

Gene amplification plays a critical role in tumor progression. Hence, understanding the factors triggering this process in human cancers is an important concern. Unfortunately, the structures formed at early stages are usually unavailable for study, hampering the identification of the initiating events in tumors. Here, we show that the region containing the PIP gene, which is overexpressed in 80% of primary and metastatic breast cancers, is duplicated in the breast carcinoma cell line T47D. The two copies are organized as a large palindrome, lying 'in loco' on one chromosome 7. Such features constitute the landmark of the breakage-fusion-bridge (BFB) cycle mechanism. In hamster cells selected in vitro to resist cytotoxic drugs, common fragile site (CFS) activation has been shown to trigger this mechanism. Here, we characterize FRA7I at the molecular level and demonstrate that it lies 2 Mb telomeric to the PIP gene and sets the distal end of the repeated sequence. Moreover, our results suggest that the BFB process was frozen within the first cycle by healing of the broken chromosome. T47D cells thus offer a unique opportunity to observe the earliest products of the BFB cycle mechanism. Our findings constitute the first evidence that this amplification mechanism can be initiated in vivo by fragile site activation.

Characterization of a Novel and Functional Human Prolactin Receptor Isoform (ΔS1PRLr) Containing Only One Extracellular Fibronectin-Like Domain

Prolactin (PRL)-dependent signaling occurs as the result of ligand-induced homodimerization of the PRL receptor (PRLr). To date, short, intermediate, and long human PRLr isoforms have been characterized. To investigate the expression of other possible human PRLr isoforms, RT-PCR was performed on mRNA isolated from the breast carcinoma cell line T47D. A 1.5-kb PCR fragment was isolated, subcloned, and sequenced. The PCR product exhibited a nucleotide sequence 100% homologous to the human long isoform except bp 71-373 were deleted, which code for the S1 motif of the extracellular domain. Therefore, this isoform was designated the deltaS1 PRLr. Northern analysis revealed variable deltaS1 PRLr mRNA expression in a variety of tissues. Transfection of Chinese hamster ovary cells with deltaS1 cDNA showed the isoform is expressed at the protein level on the cell surface with a molecular mass of approximately 70 kDa. Kinetic studies indicated the deltaS1 isoform bound ligand at a lower affinity than wild-type receptor. The deltaS1 PRLr was also shown to activate the proximal signaling molecule Jak2 upon addition of ligand to transfected cells, and, unlike the long PRLr, high concentrations of ligand did not function as a self-antagonist to signaling during intervals of PRL serum elevation, i.e. stress and pregnancy. Given its apparent widespread expression, this PRLr isoform may contribute to PRL action. Furthermore, the functionality of this receptor raises interesting questions regarding the minimal extracellular domain necessary for ligand-induced receptor signaling.

PLIF, a Novel Human Ferritin Subunit from Placenta with Immunosuppressive Activity

Ferritin is a ubiquitous iron storage protein existing in multiple isoforms composed of 24 heavy and light chain subunits. We describe here a third ferritin-related subunit cloned from human placenta cDNA library and named PLIF (placental immunomodulatory ferritin). The PLIF coding region is composed of ferritin heavy chain (FTH) sequence lacking the 65 C-terminal amino acids, which are substituted with a novel 48 amino acid domain (C48). In contrast to FTH, PLIF mRNA does not include the iron response element in the 5'-untranslated region, suggesting that PLIF synthesis is not regulated by iron. The linkage between the FTH and C48 domains created a restriction site for EcoRI. PLIF protein was found to localize in syncytiotrophoblasts of placentas (8 weeks of gestation) at the fetal-maternal interface. Increased levels of PLIF transcript and protein were also detected in the breast carcinoma cell lines T47D and MCF-7 but not in the benign corresponding cell line HBL-100. In vitro, PLIF was shown to down-modulate mixed lymphocyte reactions and to inhibit the proliferation of peripheral blood mononuclear cells stimulated with OKT3. The accumulated data indicate that PLIF is an embryonic immune factor involved in down-modulating the maternal immune recognition of the embryo toward anergy. This mechanism may have been adapted by breast cancer cells over expressing PLIF.

Testosterone and 5α-dihydrotestosterone inhibit in vitro growth of human breast cancer cell lines

Androgens are of biological and clinical importance for the growth and development of breast cancer in women, and the androgen receptor (AR) has been shown to be a predictor of tumor differentiation. Inthe present study, we investigated the relationship between AR status and testosterone and 5α-dihydrotestosterone (DHT)-dependent proliferation of the human breast carcinoma cell lines MCF-7,T47-D, MDA-MB 435S and BT-20. AR status was studied by means of immunocytochemistry and Western blot analysis. All four cell lines stained positively for AR. Western blot analysis revealed a strong expressionof AR in MCF-7, in contrast to BT-20 cells. According to proliferation kinetics, we observed a significant (p ≤ 0.05) dose-dependent inhibition of cell growth by testosterone and DHT treatment in allfour cell lines. In the estrogen receptor (ER)-negative cell lines BT-20 and MDA-MB 435S, testosterone was a more potent inhibitor of cell proliferation than DHT (p ≤ 0.05), in contrast to the ER-positivecells lines MCF-7 and T47-D, in which a stronger inhibition of proliferation was achieved by DHT. A partial transformation of testosterone to estrogen in ER-positive cells might be an explanation for thiseffect. Our data favor a possible role of androgens in growth regulation of breast cancer. Clinical studies are needed to analyze the importance of AR as a possible predictor in response to endocrine therapyof breast cancer.

An Essential Role for Src Kinase in ErbB Receptor Signaling through the MAPK Pathway

ErbB receptor tyrosine kinases are activated by multiple ligands such as epidermal growth factor (EGF) and neuregulins (NRGs), leading to stimulation of intracellular signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade. We show here that Src kinase is essential for rapid EGF- and NRG-induced MAPK activation when the breast carcinoma cell lines T47D and SKBR3 are stimulated with low concentrations of ligand. In the presence of the pharmacological inhibitor CGP77675, which specifically blocks the activity of Src family kinases, ligand-induced MAPK activation was almost completely blocked at 5 min. Although this block was only transient, inactivation of Src suppressed ligand-induced transcription from a MAPK-responsive promoter. At the molecular level, the initial inhibition of MAPK by Src inactivation correlated with impaired ligand-induced Shc phosphorylation. Surprisingly, Src inhibition affected neither association of Shc with ErbB receptors nor phosphorylation of receptor-bound Shc. Thus, ErbB signaling requires the engagement of a novel Src-dependent route to MAPK, to trigger its rapid activation and subsequent efficient stimulation of transcription.

A STAT-responsive Element in the Promoter of the Episialin/MUC1 Gene Is Involved in Its Overexpression in Carcinoma Cells

The mucin-like glycoprotein episialin (MUC1) is highly overproduced by a number of human carcinomas. We have shown previously in a variety of mammalian cell lines that overexpression of this very large transmembrane molecule diminishes cellular adhesion, suggesting that episialin/MUC1 overexpression may play an important role in tumor invasion and metastasis. By using in situ hybridization, we show here that episialin/MUC1 mRNA expression can be increased more than 10-fold in breast carcinoma cells relative to the expression in adjacent normal breast epithelium. In search of the molecular mechanism of this overexpression, we observed that the episialin/MUC1 promoter contains a candidate binding site for transcription factors of the STAT family approximately 500 base pairs upstream of the transcription start site. Cytokines and/or growth factors such as interleukin-6 or interferon-gamma can activate STATs. In the human breast carcinoma cell line T47D, both compounds are able to stimulate transcription of a luciferase reporter gene under the control of a 750-base pair MUC1 promoter fragment proximal to the transcription start site. The observed increase is entirely mediated by the single STAT-binding site, since mutation of this site abolishes stimulation of the reporter by interleukin-6 and interferon-gamma. In addition, mutation of the STAT site also decreased the promoter activity in nonstimulated T47D cells, suggesting that the STAT-binding site is among the elements that are involved in the overexpression of MUC1 in tumor cells.

Downregulation of VPAC1R expression in breast cancer cell lines.

The breast carcinoma cell line T47D was tested for 17 beta-estradiol (E2) mediated regulation of vasoactive intestinal polypeptide receptor type-1 (VPAC1) expression. E2 was found to downregulate the mRNA level. The number of VIP binding sites was reduced 66% on treatment with E2 for 72 h. Experiments with cycloheximide suggested that the effect was independent (at least partly so) of protein synthesis. Experiments with the transcriptional inhibitor, actinomycin D, showed that E2 did not influence the VPAC1 mRNA halflife. Both of two antiestrogens, ICI 182,780 and 4-hydroxy-tamoxifen, mediated a concentration dependent inhibition of the effect of E2 on the mRNA level. Transient transfection with reporter-gene constructs containing various portions of the VPAC1 5'-flanking sequence revealed the most proximal 100 bp to be essential for the basal transcriptional activity. However, E2 did not influence the expression of the reporter gene using up to 3,250 bp of the VPAC1 5'-flanking region.